Comparison of monomeric and tetrameric antigen for identification of memory B cells. (A) Mono-biotinylated TTCF or CD80 antigens were directly labeled with the Alexa-488 fluorophore; tetramers were generated with unlabeled streptavidin. Enriched B cells from each donor were split into 3 fractions and stained with control CD80 tetramer, TTCF monomer, or TTCF tetramer at the same total antigen concentration of 0.125 μg/mL. Fluorescence-activated cell sorter plots depict CD19⁺ CD27⁺ IgM⁻ class-switched memory B cells; numbers adjacent to the gate represent the percentage of the parental gate. (B) Frequencies of tetramer⁺ memory B cells detected in 3 different donors. Numbers are calculated as tetramer-positive cells per 1 × 10⁶ CD19⁺ memory B cells.